We have been studying the molecular mechanisms by which the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), via thyroid hormone nuclear receptors (TRs), promotes growth, differentiation and development. (a) To understand how T3 stimulates cell growth, we analyzed the effect of T3 on the progression of cell cycle in rat pituitary GC101 cells. T3 stimulated cell growth by reducing the doubling time about 2-fold. This stimulatory effect was mainly due to the shortening of G0/G1 phase. The kinase activities associated with cyclins D1 and E, the two key regulators in G1 to S transition, were activated 6-8 fold by T3. This activation was due to not only the increase in the expression of both cyclins at the protein level but also of CDK2. The T3-induced increase in CDK activities resulted in the hyperphosphorylation of the retinoblastoma protein (Rb). These findings identified for the first time the pathways via which T3 mediated the proliferation of cells. The functional link of TRs to Rb has important implications in the understanding of the biology of normal and cancer cells. (b) To delinate molecular pathways of nuclear translocation and targeting of TRs to the regulatory sites in chromatin, we prepared green fluorescent protein conjugated to the human TR, subtype beta 1 (GFP-TR beta 1). Using high- resolution video and confocal scanning microscopy and living cells, we found that in the presence of T3, most of the transfected GFP-TR beta 1 was localized in the nucleus, excluding nucleoli. In the absence of T3, GFP-TR beta 1 was evenly distributed between the nucleus and cytoplasm. The GFP-TR beta 1 which was in the cytoplasm could be induced to translocate into the nucleus by T3. This is the first demonstration of T3-induced cytoplasm-nucleus trafficking which provides a novel regulatory role of T3 on TR action. (c) It is unclear whether the two TR isoforms, TR alpha 1 and TR beta 1, mediate differential biological activities. It is known that mutations of TR beta gene lead to a human disease known as thyroid hormone resistance syndrome; whereas, the transgenic mice expressing a mutated form of TR alpha 1 (v-erbA) develop hepatocellular carcinoma. We postulated that TR alpha mutants may associate with hepatocellular carcinoma. To test this hypothesis, we isolated and sequenced the cDNAs from tumor tissues of patients with hepatocellular carcinoma. Two TR alpha mutants (TR alpha 1-H, V390A; TR alpha 1-L:E350K and P398S) were identified which lacked T3 binding and transcriptional activities. More importantly, these two mutants interfered with the normal functions of TRs via dominant negative action. These results clearly indicate that the TR alpha and TR beta mediate distinct biolgoical functions. Furthermore, these findings support the hypothesis that TR alpha mutants may contribute to the development of hepatocellular carcinoma.